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于2007年3月,采集大连大窑湾浮筏贝类养殖区的太平洋牡蛎,抽取牡蛎的血淋巴液进行沿岸单孢子虫的研究。采用显微镜观察、聚合酶链式反应(PCR)和原位杂交(ISH)方法对血淋巴液样品进行检测。结果在显微镜下观察到牡蛎血淋巴液中存在沿岸单孢子虫(Haplosporidium costale,SSO)原生质体的类似物;提取血淋巴液基因组DNA,应用扩增沿岸单孢子虫SSUrDNA区域的引物对进行PCR扩增,产生约150bp的基因片段,经测序并将测序结果与基因库中已知序列比对分析,确定这种类似物为沿岸单孢子虫。同时,采用沿岸单孢子虫的特异性DNA探针SSO1318,对牡蛎血淋巴液中的沿岸单孢子虫进行原位杂交,结果为阳性。
In March 2007, Pacific oysters were collected from the aquaculture area of Dayan Bay in Dalian, and the haemolymph of oyster was collected for the study of Monochamosporiasis along the shore. The samples of hemolymph were detected by microscopy, polymerase chain reaction (PCR) and in situ hybridization (ISH). Results The homolog of Haplosporidium costale (SSO) protoplasts was observed in the hemolymph of oyster under the microscope. The genomic DNA of hemolymph was extracted and amplified by PCR using primer pairs of SSUrDNA region of S. monocytogenes An approximate 150bp gene fragment was generated and sequenced. The sequencing results were aligned with known sequences in the gene bank to confirm that the analogue was S. parasuis. At the same time, in situ hybridization of Monochamus spp. In the haemolymph of oyster was carried out by using the specific DNA probe SSO1318 of Monosialosporium along the coast, and the result was positive.